[The ketogenic diet: an underappreciated therapeutic option?]. / La dieta chetogenica: un'opportunità terapeutica ignorata?

Obesity is reaching epidemic proportions in Western countries and is a strong risk factor for cardiovascular disease. Despite the constant recommendations of health care organizations regarding the importance of weight control, this goal often fails. Although there is a common agreement about the concept that exercise and diet are two key factors for the control of body weight, the ideal amount and type of exercise and also the ideal diet for weight control are still under debate. A widely accepted nutritional regime is the Mediterranean diet that has evident health benefits although less attention has been paid to see if the effects are due to other lifestyle factors which may contribute to the health benefits perhaps as much as specific food choices. There are several other options available to the physician that may produce good weight loss results in the short/medium term and also for maintenance of the goal achieved. One of these strategies is the ketogenic diet or VLCKD (very low carbohydrate ketogenic diet) that has been widely studied in recent years. Most studies show that this diet has a solid physiological and biochemical basis which is able to induce effective weight loss and improvement of several parameters of cardiovascular risk. This review discusses the physiological basis of VLCKD and the main applications together with its strengths and weaknesses compared to common dietary recommendations.

Sex steroid receptors, secondary bile acids and colorectal cancer. A possible mechanism of interaction.

AIM: The aim of the work was to study in colon-rectum cancer mucosae the binding charateristics, as sex steroid receptors. METHODS: Specific androgen (AR), estrogen (ER) and progesterone (PgR) receptors were measured in the tissue samples of 35 patients (15 males, 20 females) undergoing colectomy or coloproctectomy for adenocarcinoma. The characteristics of androgen receptor (AR, DHT-R: dihydrotestosterone receptor) were also investigated using competitive activity of cyproterone acetate, cortisol, aldosterone and steroid-like substances such as deoxycholic and lithocholic acid, present in the milieu of the considered organ. Binding assays and competition tests were conducted using a charcoal dextran method. RESULTS: When present (50%), ER and PgR receptors showed very low levels and no difference was noted between cancerous and the surrounding healthy mucosa. AR were found in all samples from both neoplastic and non neoplastic surrounding mucosa, with no significant difference. Androgen receptor however exhibited an altered binding activity in cancer specimens. Cyproterone acetate did not displace DHT from AR while significant displacing activity was elicited by DHT, testosterone, as well as by lithocholic acid, but not by deoxycholic acid. CONCLUSION: In cancerous large bowel mucosa, androgen receptors show altered binding characteristics. The selective binding of lithocholic acid to AR supports the hypothesis that diet-related endoluminal substances may play a role in cancer development model where molecular alterations such as DNA damage or mutation is the 1st event.

The control of progesterone receptor expression in MCF-7 breast cancer cells: effects of estradiol and sex hormone-binding globulin (SHBG).

Estradiol controls the gene transcription and expression of many proteins in breast cancer cells, like the progesterone receptor, PR, that is up-regulated by the hormone. Moreover, estradiol is one of the crucial factors inducing the proliferation of breast cancer cells. Sex Hormone-Binding Globulin (SHBG), the plasma carrier for both estradiol and androgens, inhibits the estradiol-induced growth of MCF-7 cells (estrogen-dependent breast cancer cells), through its membrane receptor (SHBG-R), cAMP and PKA. The anti-estrogenic effect of SHBG, which has been described only as far as cell proliferation is concerned, could also play a meaningful role in the estradiol control of other factors in breast cancer cells. In the present study, the effect of SHBG on the estradiol control of PR expression (both mRNA and protein) and function (receptor binding capacity) in MCF-7 cells was examined. SHBG inhibited the estradiol-induced up-regulation of PR mRNA as well as protein level and function. Moreover, the effect of SHBG on estradiol control of PR expression and function was showed to be specific and mediated by PKA. The intermediacy of PKA in the PR expression control, together with the observation that it is effective in the condition in which the SHBG receptor is activated, supports the hypothesis that the anti-estrogenic effect of SHBG could be receptor-mediated. The ability of SHBG to inhibit estradiol action in a specific way in estrogen-dependent breast cancer cells has, therefore, to be taken into account for the development of future therapeutic strategies.

Estradiol induction of cAMP in breast cancer cells is mediated by foetal calf serum (FCS) and sex hormone-binding globulin (SHBG).

Plasma sex hormone-binding globulin (SHBG or SBP), the specific carrier for estradiol and androgens, after binding to its membrane receptor (SHBG-R), causes a significant increase of cAMP in the presence of estradiol, in both breast (MCF-7) and prostate (LNCaP) cancer cells maintained in serum-free medium. On the other hand, it has been proposed that estrogens, in addition to the well-known nuclear receptor pathway, exert their biological effect inducing cAMP, as a consequence of a direct membrane action, in breast cancer and uterine cells. The aim of the present study was to clarify this controversial issue by verifying if the cAMP increase in MCF-7 cells was a direct effect of estradiol, or if it was mediated by FCS proteins, such as bovine sex hormone-binding globulin; and to reevaluate the effect of human SHBG on cAMP induction in the presence of FCS. MCF-7 cells were maintained in DCC-FCS (treated with DCC to remove steroids), in SHBG-FREE/DCC-FCS (treated with DCC and with a specific affinity chromatography to remove bovine sex hormone-binding globulin), or in serum-free medium (SFM). It was observed that estradiol determined a significant time-dependent increase of cAMP only in MCF-7 cells maintained in 10% DCC-FCS. When cells were maintained in 10% SHBG-FREE/DCC-FCS, estradiol had no detectable effect. However, its ability to increase cAMP was observed again after the addition of human SHBG, in doses ranging from 5 to 50 nM. Moreover, in the presence of 10% SHBG-FREE/DCC-FCS, SHBG, even in the absence of estradiol, caused a significant increase of cAMP. In conclusion, the data reported in the present study suggest that the ability of estradiol to induce cAMP in MCF-7 cells is not due to a direct membrane effect of the hormone, but rather it is mediated by FCS. SHBG is one of the serum factors mediating estradiol action. Lastly, it was proven that SHBG triggers the cAMP pathway in MCF-7 cells in a physiologic culture condition and at physiologic concentrations.

The additionally glycosylated variant of human sex hormone-binding globulin (SHBG) is linked to estrogen-dependence of breast cancer.

Sex Hormone-Binding Globulin (SHBG), the plasma carrier for androgens and estradiol, inhibits the estradiol-induced proliferation of breast cancer cells through its membrane receptor, cAMP, and PKA. In addition, the SHBG membrane receptor is preferentially expressed in estrogen-dependent (ER+/PR+) breast cancers which are also characterized by a lower proliferative rate than tumors negative for the SHBG receptor. A variant SHBG with a point mutation in exon 8, causing an aminoacid substitution (Asp 327-->Asn) and thus, the introduction of an additional N-glycosylation site, has been reported. In this work, the distribution of the SHBG variant was studied in 255 breast cancer patients, 32 benign mammary disease patients, and 120 healthy women. The presence of the SHBG mutation was evaluated with PCR amplification of SHBG exon 8 and Hinf I restriction fragment length polymorphism (RFLP) procedure. This technique allowed us to identify 54 SHBG variants (53 W/v and 1 v/v) in breast cancer patients (21.2%), 5 variants (4 W/v and 1 v/v) in benign mammary disease patients (15.6%), and 14 variants (W/v) in the control group (11.6%). The results of PCR and RFLP were confirmed both by nucleotide sequence of SHBG exon 8 and western blot of the plasma SHBG. No differences in the mean plasma level of the protein were observed in the three populations. The frequency of the SHBG variant was significantly higher in ER+/PR+ tumors and in tumors diagnosed in patients over 50 years of age than in the control group. This observation suggests the existence of a close link between the estrogen-dependence of breast cancer and the additionally glycosylated SHBG, further supporting a critical role of the protein in the neoplasm.

Prolactin is an autocrine growth factor for the Jurkat human T-leukemic cell line.

Despite convincing evidence of cooperation between IL-2 and endogenous prolactin (PRL) during T cell activation, the individual role of PRL as a T-cell lineage cytokine remains to be defined. We have examined the production and function of PRL on the Jurkat human T-leukemic cell line, which does not constitutively produce IL-2. The majority of Jurkat cells expressed PRL receptor (R) under standard culture conditions, whereas appearance of the alpha chain of the IL-2-R required PHA-PMA stimulation, as did IL-2 synthesis. Western blotting revealed a predominant band at 23.5 kDa and a weaker band at 25.5 kDa in both Jurkat cell lysates and human (h) pituitary PRL. Metabolic labeling of the cell lysates with 35S-methionine and immunoprecipitation with an antiserum against hPRL showed that both forms of PRL are actively synthesized by the Jurkat cell line. PRL released in the medium was biologically active in the rat Nb2 lymphoma mitogenic assay. Depletion of medium PRL with two polyclonal anti-hPRL antisera inhibited the growth of Jurkat cells in a dose-dependent manner, as evaluated by cell number and 3H-TdR uptake. Purified pituitary or recombinant hPRL at a wide range of concentrations had no significant effect on their growth, but reversed the blocking activity of the anti-hPRL antibody. Recombinant IL-2 had no effect on the antibody-induced growth inhibition. Taken as a whole, these results demonstrate that PRL can act as an autocrine T cell growth factor independently of IL-2 and are the first evidence of its involvement in human leukemic growth and possibly in leukemic transformation.

Sex steroid binding protein receptor (SBP-R) is related to a reduced proliferation rate in human breast cancer.

In the last years, an increasing amount of studies described a membrane receptor for the Sex Steroid Binding Protein (SBP) on several androgen-estrogen dependent tissues. One of the suggested biological roles of the interaction between SBP and its receptor seems to be a negative control of the E2 induced proliferation of human breast cancer cells through the cAMP pathway. In the present work, SBP membrane receptor was evaluated on human breast cancer specimens with a radio-binding assay. Each tissue sample was also evaluated for ER and PGR status. Cytosol Thymidine Kinase levels were measured in tissue samples in order to evaluate cell proliferation rate. SBP binding to membranes of ER +/PGR + samples was time and temperature dependent, specific and at high affinity. In addition, SBP recognized on breast cancer membranes two sites at different affinity, as previously described for other human tissues and cultured cells. Membrane SBP-R was detected in a significantly higher number of samples positive for both ER and PGR than in negative samples. SBP-R positive samples showed a significantly lower proliferation rate than SBP-R negative samples as demonstrated by TK activity. The present study contains evidences for the existence of a specific membrane receptor for SBP in breast cancer sample membranes and the presence of SBP-R seems to be strictly related to a lower proliferation rate of the sample.

Sex steroid binding protein exerts a negative control on estradiol action in MCF-7 cells (human breast cancer) through cyclic adenosine 3',5'-monophosphate and protein kinase A.

Estradiol is considered to be a critical factor in the growth induction of some breast cancer cells, like MCF-7 cell line. Among other compounds involved in the control of neoplastic mammary cell growth, cAMP has been suggested, on the other hand, to exert an antiproliferative effect. Sex steroid binding protein (SBP) sex hormone binding globulin (SHBG), the plasma carrier for both androgens and estradiol, recognizes a specific receptor located on membranes of estrogen- and androgen-sensitive tissue and cultured cells (e.g. MCF-7 cell). The interaction of estradiol with the receptor-bound SBP has been reported to induce a significant accumulation of cAMP in MCF-7 cells; in addition, a negative modulation of estradiol induced proliferation of these cells has been described after treatment with SBP. We report here a more detailed observation about the effect of SBP on MCF-7 cell estradiol-induced growth as well as the possible linkage between SBP and its membrane receptor and protein kinase A activity. MCF-7 cell growth was induced by estradiol, but the effect of estradiol was completely abolished by cell treatment with both SBP and estradiol. The inhibitory effect of SBP was highly specific. Because it was suggested that SBP might act through cAMP, we investigated the effect of SBP and estradiol in cells treated with protein kinase A inhibitor peptide (6-22) amide, a specific inhibitor of the cAMP target protein kinase A. The blockade of PKA had no effect on estradiol action on cell growth but masked completely the effect of SBP because MCF-7 increased growth sustained by estradiol was fully detectable also in the presence of SBP. We also observed that MCF-7 cells treated with increasing doses of 8Br-cAMP, cAMP analog and PKA activator, showed a progressive reduction of their growth. 8Br-cAMP was also able to inhibit estradiol promotion of MCF-7 cell growth. The inhibitory effect of 8Br-cAMP on estradiol-induced proliferation was already detectable at analog concentration of 100 nM, which has been reported to be the level reached by cAMP in MCF-7 cells treated with SBP and estradiol. In conclusion, the present study strongly confirms our previous observation that SBP inhibits the estradiol induction of MCF-7 cell growth, appropriately suggesting that this SBP action, a consequence of the interaction with the receptor, is likely to be mediated by cAMP and PKA. In addition, the study implies a significant role of cAMP in the control of breast cancer cell growth.

Sex steroid-binding protein and its membrane receptor in estrogen-dependent breast cancer: biological and pathophysiological impact.

Data obtained in our laboratory about the membrane receptor for sex steroid-binding protein (SBP-R) in human breast cancer are reported. SBP-R was detected in MCF-7 cells (estrogen receptor positive, ER+), while MDA-MB 231 cells (ER-) did not bind SBP. MCF-7 cells treated with SBP and E2 showed a marked increase of intracellular cAMP, and a significant reduction of both E2 induced cell proliferation and E2-mediated increase of progesterone receptor (PGR). The inhibition of E2 effects in MCF-7 cells was shown to be highly specific for SBP and mediated by protein kinase A, the target of cAMP. Membrane SBP-R was also evaluated in primary breast cancers. SBP-R was detectable only on ER+/PR+ samples and SBP-R+ samples presented a lower proliferation rate than negative samples. Our data, thus suggest that SBP-R and ER could be functionally related and also that SBP could modulate estrogen action at target cell site.

The receptor-mediated action of sex steroid binding protein (SBP, SHBG): accumulation of cAMP in MCF-7 cells under SBP and estradiol treatment.

The interaction of sex steroid binding protein (SBP) with its specific receptor in MCF-7 cell (estrogen-sensitive human breast cancer cells), followed by the binding of estradiol (E2) to the complex SBP-receptor, induced a significant accumulation of intracellular cAMP. SBP alone as well as E2 alone did not elicit any modification of the nucleotide. The maximal increase in cAMP was observed with 1 nM SBP + 1 nM E2. Increasing doses of both SBP and E2, even raising cAMP levels with respect to basal, did not give any higher response. Both testosterone and dihydrotestosterone, used instead of E2, were not able to induce any significant modification of cAMP. E2-induced MCF-7 cell proliferation was significantly reduced by 8Br-cAMP. MDA-MB 231 cells (estrogen-insensitive breast cancer cells) were not shown to bind SBP, or to respond to SBP + E2 as far as both their proliferation and cAMP content are concerned. In summary, the present study provides evidence that the SBP receptor is part of the G-protein receptor family, and that SBP can act as modulator of E2 action at cell site through the second messenger cAMP.

Hormonal and clinic evaluation of patients with moderate body hair growth.

Plasma prolactin (PRL), gonadotropins (FSH, LH), estradiol-17 beta (E2), progesterone (P), total testosterone (T), sex steroid binding protein (SBP), T/SBP index, cortisol (F), 17-OH-progesterone (17OH-P), dehydroepiandrosterone sulphate (DHEA-S) and androstenedione (A), were measured in 50 fertile non-obese women presenting with moderate body hair growth and in 30 matched controls. DHEA-S and PRL were significantly higher (P < 0.002, P < 0.001, respectively) and SBP was lower (P < 0.001) in patients than in controls. Regression analyses showed that PRL levels were independent of the other parameters, while a negative correlation was found between DHEA-S and SBP values. Since the decision to treat a woman with mild body hair growth is usually a clinical one, PRL behaviour has to be taken into account before deciding the type of treatment. Clinical improvement was observed in subjects treated with ethynylestradiol plus desogestrel or plus cyproterone acetate, so as to produce an increase in SBP rather than a decrease in DHEA-S.

Biological relevance of the interaction between sex steroid binding protein and its specific receptor of MCF-7 cells: effect on the estradiol-induced cell proliferation.

The human breast cancer cells MCF-7 were shown to bind sex steroid binding protein (SBP) at a receptor site. The binding to whole cells was specific, time-dependent, saturable, and at high affinity. Estradiol, bound to SBP, induced a significant inhibition of SBP-cell binding at a dose of 10(-9) M. The presence of SBP, bound either to estradiol, or to cells, did not alter the amount of estradiol entering cells, but it "captured" an additional quantity of the hormone at the outer surface of cells. Furthermore, the effect of SBP on estradiol-induced MCF-7 cell proliferation was evaluated. While estradiol is an effective proliferating agent on MCF-7 cells, SBP itself did not produce any significant cell proliferation; the growth of MCF-7 cells in the presence of the complex SBP-estradiol was not different from the growth in the presence of estradiol alone; SBP bound to its receptor produced a significant reduction of the estradiol-induced cell proliferation. In summary, the present study provides evidence that the interaction of SBP with its receptor on MCF-7 cells is not involved in the uptake of estradiol, but it can modify the effect of estradiol at target site by a mechanism which is not likely to be a simple sequestration of the hormone at the outer surface of cells.

The membrane receptor for sex steroid binding protein is not ubiquitous.

The tissue distribution of the membrane receptor for the Sex Steroid Binding Protein (SBP) has been studied, either in estrogen/androgen dependent tissues and in tissues not strictly sex steroid dependent. A specific interaction of SBP with cell membranes has been observed to occur only in estrogen/androgen dependent tissues, some of them had been previously shown by our laboratory and by other authors to possess a specific receptor for the protein. Thus, the sex steroid dependence of the tissue is likely to be determinant for the expression of the membrane receptor for Sex Steroid Binding Protein.

Receptor for sex steroid-binding protein of endometrium membranes: solubilization, partial characterization, and role of estradiol in steroid-binding protein-soluble receptor interaction.

The sex steroid-binding protein (SBP) receptor was solubilized from the membranes of human premenopausal endometrium with the zwitterionic detergent CHAPS. The binding activity of the soluble receptor was studied, allowing it to interact with [125I]SBP and precipitating the complex with polyethylene glycol 8,000. The interaction of SBP with the soluble receptor was specific, saturable, and at high affinity. Indeed, the specific binding was definitely improved on the solubilized form of the receptor. The effect exerted by sex steroids on the interaction of SBP with receptor was also examined on both the soluble and membrane-bound forms. At physiologic doses (10(-8) M) estradiol inhibits the binding at a significant extent on the soluble receptor, but not on membrane-bound form. The dose of estradiol required to significantly inhibit the SBP-specific binding was dependent on the form of receptor. In membrane-bound receptor the inhibiting dose of estradiol was higher than its physiologic concentration. Thus, it is likely that, while soluble receptor cannot recognize the complex steroid-SBP, membrane-bound receptor can interact both with "unliganded" SBP and with the estradiol-SBP complex (but not with androgen-SBP complexes) in an estrogen-dependent tissue like human endometrium.

The receptor for human sex steroid binding protein (SBP) is expressed on membranes of neoplastic endometrium.

Sex steroid binding protein (SBP) receptor was detected on cell membranes obtained from human endometrium adenocarcinoma. The binding of SBP was proved to be highly specific, saturable, and at high affinity. It was, additionally, shown to occur at two sites at different affinities, as previously described for other human tissues. SBP was, therefore, demonstrated to recognized a specific receptor on endometrium adenocarcinoma membranes. The effect of steroid hormones on SBP-receptor interaction was also evaluated. Both dihydrotestosterone and estradiol were shown to inhibit the binding of SBP to its specific receptor on neoplastic membranes. Testosterone at a dose of 10(-9) M was shown not to interfere to a significant extent with SBP-receptor binding. The sensitivity for estradiol we had previously observed in normal premenopausal endometrium was completely lost in postmenopausal neoplastic tissue. These observations suggest that the SBP-membrane recognition system is still present in neoplastic postmenopausal endometrium, but it has been modified either by the postmenopausal endogenous milieu or by the neoplastic transformation.

Smoking effects on the hormonal balance of fertile women.

We evaluated serum pituitary hormones (prolactin, follicle-stimulating hormone, luteinizing hormone), gonadal hormones (estrone, estradiol, progesterone), sex steroid binding protein (SBP) and urine estrogens in 684 healthy fertile women, subdivided into smokers (n = 237) and nonsmokers (n = 447). The aim of the work was to elucidate whether smoking habits can affect hormonal balance. Smoking interference of estrogen metabolism has been postulated, but no unequivocal data have been reported. A protective role against breast cancer has even been suggested on the basis of a reduced estrogenic activity found in smokers. Our data showed a considerable interference of smoking on PRL secretion, probably related to a direct inhibiting activity of nicotine. Estrogen catabolism could also be involved, and a catabolic shift of 16 alpha-hydroxylation in favour of 2 alpha-hydroxylated catabolites, via the hepatic cytochrome P-450 system could be hypothesized.

Sex steroid-binding protein interacts with a specific receptor on human premenopausal endometrium membrane: modulating effect of estradiol.

Sex steroid-binding protein receptor was detected on membranes prepared from human premenopausal endometrium. The binding of sex steroid-binding protein to membranes was specific, saturable, and high affinity. Scatchard analysis showed the presence of two binding sites at different affinities. The addition of estradiol (10(-8) M) did not produce any inhibition of binding; indeed, it resulted in a modification of binding characteristics. The demonstration of sex steroid-binding protein receptor on membranes of human premenopausal endometrium indicates that the expression of receptor on membranes is not an effect of estrogen over stimulation on target tissues. Estradiol could act as a modulating factor of the binding, probably reflecting the sensitivity of tissues to different steroids.

Sex steroid binding protein (SBP) receptors in estrogen sensitive tissues.

Since the discovery of a specific membrane binding site for sex steroid binding protein (SBP) in human decidual endometrium and in hyperplastic prostate numerous speculations have been raised on the existence of an additional non-receptor-mediated system for steroid hormone action. In the present work SBP cell membrane binding was investigated in human estrogen target tissues other than those previously studied either in the absence of steroids or in the presence of varying amounts (10(-10)-10(-6) M) of estradiol, testosterone and dihydrotestosterone, respectively. Plasma membranes obtained by differential centrifugation from homogenized samples of pre-menopausal endometrium, endometrium adenocarcinoma, normal liver and post-menopausal breast showed a specific binding of highly purified [125I]SBP: a major displacement of labeled SBP was elicited by radioinert SBP, while no significant displacement occurred when other human plasma proteins were used as cold competitors (molar excess ranging 500-10,000-fold). A specific, time-dependent binding of [125I]SBP was also observed in MCF-7 and in Hep-G2 cell lines. The different patterns of specific binding, observed in membranes from different tissues when SBP was liganded with different sex steroid molecules, leads us to consider the tissue individuality of the receptor as a further entity in the membrane recognition system for SBP.